Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors

ABSTRACT

The present invention provides a process for growing eukaryotic microorganisms which are capable of producing lipids, in particular lipids containing polyenoic fatty acids. The present invention also provides a process for producing eukaryotic microbial lipids.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority under 35U.S.C. §119(e) from Provisional Patent Application Serial No.60/178,588, filed on Jan. 28, 2000. Provisional Patent ApplicationSerial No. 60/178,588 is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

[0002] The present invention is directed to a novel process for growingmicroorganisms and recovering microbial lipids. In particular, thepresent invention is directed to producing microbial polyunsaturatedlipids.

BACKGROUND OF THE INVENTION

[0003] Production of polyenoic fatty acids (fatty acids containing 2 ormore unsaturated carbon-carbon bonds) in eukaryotic microorganisms isgenerally known to require the presence of molecular oxygen (i.e.,aerobic conditions). This is because it is believed that the cis doublebond formed in the fatty acids of all non-parasitic eukaryoticmicroorganisms involves a direct oxygen-dependent desaturation reaction(oxidative microbial desaturase systems). Other eukaryotic microbiallipids that are known to require molecular oxygen include fungal andplant sterols, oxycarotenoids (i.e., xanthophyls), ubiquinones, andcompounds made from any of these lipids (i.e., secondary metabolites).

[0004] Eukaryotic microbes (such as algae; fungi, including yeast; andprotists) have been demonstrated to be good producers of polyenoic fattyacids in fermentors. However, very high density cultivation (greaterthan about 100 g/L microbial biomass, especially at commercial scale)can lead to decreased polyenoic fatty acid contents and hence decreasedpolyenoic fatty acid productivity. This may be due in part to severalfactors including the difficulty of maintaining high dissolved oxygenlevels due to the high oxygen demand developed by the high concentrationof microbes in the fermentation broth. Methods to maintain higherdissolved oxygen level include increasing the aeration rate and/or usingpure oxygen instead of air for aeration and/or increasing the agitationrate in the fermentor. These solutions generally increase the cost oflipid production and can cause additional problems. For example,increased aeration can easily lead to severe foaming problems in thefermentor at high cell densities and increased mixing can lead tomicrobial cell breakage due to increased shear forces in thefermentation broth (this causes the lipids to be released in thefermentation broth where they can become oxidized and/or degraded byenzymes). Microbial cell breakage is an increased problem in cells thathave undergone nitrogen limitation or depletion to induce lipidformation, resulting in weaker cell walls.

[0005] As a result, when lipid producing eukaryotic microbes are grownat very high cell concentrations, their lipids generally contain onlyvery small amounts of polyenoic fatty acids. For example, the yeastLipomyces starkeyi has been grown to a density of 153 g/L with resultinglipid concentration of 83 g/L in 140 hours using alcohol as a carbonsource. Yet the polyenoic fatty acid content of the yeast atconcentration greater than 100 g/L averaged only 4.2% of total fattyacids (dropping from a high of 11.5% of total fatty acid at a celldensity of 20-30 g/L). Yamauchi et al., J. Ferment. Technol., 1983, 61,275-280. This results in a polyenoic fatty acid concentration of onlyabout 3.5 g/L and a polyenoic fatty acid productivity of only about0.025 g/L/hr. Additionally, the only polyenoic fatty acid reported inthe yeast lipids was C18:2.

[0006] Another yeast, Rhodotorula glutinus, has been demonstrated tohave a lipid productivity of about 0.49 g/L/hr, but also a low overallpolyenoic fatty acid content in its lipids (15.8% of total fatty acids,14.7% C18:2 and 1.2% C18:3) resulting in a polyenoic fatty acidproductivity in fed-batch culture of only about 0.047 g/L/hr and 0.077g/L/hr in continuous culture.

[0007] Present inventors have previously demonstrated that certainmarine microalgae in the order Thraustochytriales can be excellentproducers of polyenoic fatty acids in fermentors, especially when grownat low salinity levels and especially at very low chloride levels.Others have described Thraustochyrids which exhibit a polyenoic fattyacid (DHA, C22:6n-3; and DPA, C22:5n-6) productivity of about 0.158g/L/hr, when grown to cell density of 59 g/L/hr in 120 hours. However,this productivity was only achieved at a salinity of about 50% seawater,a concentration that would cause serious corrosion in conventionalstainless steel fermentors.

[0008] Costs of producing microbial lipids containing polyenoic fattyacids, and especially the highly unsaturated fatty acids, such asC18:4n-3, C20:4n-6, C20:5n3, C22:5n-3, C22:5n-6 and C22:6n-3, haveremained high in part due to the limited densities to which the highpolyenoic fatty acid containing eukaryotic microbes have been grown andthe limited oxygen availability both at these high cell concentrationsand the higher temperatures needed to achieve high productivity.

[0009] Therefore, there is a need for a process for growingmicroorganisms at high concentration which still facilitates increasedproduction of lipids containing polyenoic fatty acids.

SUMMARY OF THE INVENTION

[0010] The present invention provides a process for growing eukaryoticmicroorganisms which are capable of producing at least about 20% oftheir biomass as lipids and a method for producing the lipids.Preferably the lipids contain one or more polyenoic fatty acids. Theprocess comprises adding to a fermentation medium comprising eukaryoticmicroorganisms a carbon source, preferably a non-alcoholic carbonsource, and a nitrogen source. Preferably, the carbon source and thenitrogen source are added at a rate sufficient to increase the biomassdensity of the fermentation medium to at least about 100 g/L.

[0011] In one aspect of the present invention, the fermentationcondition comprises a biomass density increasing stage and a lipidproduction stage, wherein the biomass density increasing stage comprisesadding the carbon source and the nitrogen source, and the lipidproduction stage comprises adding the carbon source without adding thenitrogen source to induce nitrogen limiting conditions which induceslipid production.

[0012] In another aspect of the present invention, the amount ofdissolved oxygen present in the fermentation medium during the lipidproduction stage is lower than the amount of dissolved oxygen present inthe fermentation medium during the biomass density increasing stage.

[0013] In yet another aspect of the present invention, microorganismsare selected from the group consisting of algae, fungi, protists, andmixtures thereof, wherein the microorganisms are capable of producingpolyenoic fatty acids or other lipids which requires molecular oxygenfor their synthesis. A particularly useful microorganisms of the presentinvention are eukaryotic microorganisms which are capable of producinglipids at a fermentation medium oxygen level of about less than 3% ofsaturation.

[0014] In still another aspect of the present invention, microorganismsare grown in a fed-batch process. Moreover,

[0015] Yet still another aspect of the present invention providesmaintaining an oxygen level of less than about 3% of saturation in thefermentation medium during second half of the fermentation process.

[0016] Another embodiment of the present invention provides a processfor producing eukaryotic microbial lipids comprising:

[0017] (a) growing eukaryotic microorganisms in a fermentation medium toincrease the biomass density of said fermentation medium to at leastabout 100 g/L;

[0018] (b) providing a fermentation condition sufficient to allow saidmicroorganisms to produce said lipids; and

[0019] (c) recovering said lipids,

[0020] wherein greater than about 15% of said lipids are polyunsaturatedlipids.

[0021] Another aspect of the present invention provides a lipid recoverystep which comprises:

[0022] (d) removing water from said fermentation medium to provide drymicroorganisms; and

[0023] (e) isolating said lipids from said dry microorganisms.

[0024] Preferably, the water removal step comprises contacting thefermentation medium directly on a drum-dryer without priorcentrifugation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 is a table and a plot of various lipid productionparameters of a microorganism versus the amount of dissolved oxygenlevel in a fermentation medium.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention provides a process for growingmicroorganisms, such as, for example, fungi (including yeast), algae,and protists. Preferably, microorganisms are selected from the groupconsisting of algae, protists and mixtures thereof. More preferably,microorganisms are algae. Moreover, the process of the present inventioncan be used to produce a variety of lipid compounds, in particularunsaturated lipids, preferably polyunsaturated lipids (i.e., lipidscontaining at least 2 unsaturated carbon-carbon bonds, e.g., doublebonds), and more preferably highly unsaturated lipids (i.e., lipidscontaining 4 or more unsaturated carbon-carbon bonds) such as omega-3and/or omega-6 polyunsaturated fatty acids, including docosahexaenoicacid (i.e., DHA); and other naturally occurring unsaturated,polyunsaturated and highly unsaturated compounds. As used herein, theterm “lipid” includes phospholipids; free fatty acids; esters of fattyacids; triacylglycerols; sterols and sterol esters; carotenoids;xanthophyls (e.g., oxycarotenoids); hydrocarbons; and other lipids knownto one of ordinary skill in the art.

[0027] More particularly, processes of the present invention are usefulin producing eukaryotic microbial polyenoic fatty acids, carotenoids,fungal sterols, phytosterols, xanthophyls, ubiquinones, other compoundswhich require oxygen for producing unsaturated carbon-carbon bonds(i.e., aerobic conditions), and secondary metabolites thereof.Specifically, processes of the present invention are useful in growingmicroorganisms which produce polyenoic fatty acid(s) and for producingmicrobial polyenoic fatty acid(s).

[0028] While processes of the present invention can be used to grow awide variety of microorganisms and to obtain polyunsaturated lipidcontaining compounds produced by the same, for the sake of brevity,convenience and illustration, this detailed description of the inventionwill discuss processes for growing microorganisms which are capable ofproducing lipids comprising omega-3 and/or omega-6 polyunsaturated fattyacids, in particular microorganisms which are capable of producing DHA.More particularly, preferred embodiments of the present invention willbe discussed with reference to a process for growing marinemicroorganisms, in particular algae, such as Thraustochytrids of theorder Thraustochytriales, more specifically Thraustochytriales of thegenus Thraustochytrium and Schizochytrium, including Thraustochytrialeswhich are disclosed in commonly assigned U.S. Pat. Nos. 5,340,594 and5,340,742, both issued to Barclay, all of which are incorporated hereinby reference in their entirety. It is to be understood, however, thatthe invention as a whole is not intended to be so limited, and that oneskilled in the art will recognize that the concept of the presentinvention will be applicable to other microorganisms producing a varietyof other compounds, including other lipid compositions, in accordancewith the techniques discussed herein.

[0029] Assuming a relatively constant production rate of lipids by analgae, it is readily apparent that the higher biomass density will leadto a higher total amount of lipids being produced per volume. Currentconventional fermentation processes for growing algae yield a biomassdensity of from about 50 to about 80 g/L or less. The present inventorshave found that by using processes of the present invention, asignificantly higher biomass density than currently known biomassdensity can be achieved. Preferably, processes of the present inventionproduces biomass density of at least about 100 g/L, more preferably atleast about 130 g/L, still more preferably at least about 150 g/L, yetstill more preferably at least about 170 g/L, and most preferablygreater than 200 g/L. Thus, with such a high biomass density, even ifthe lipids production rate of algae is decreased slightly, the overalllipids production rate per volume is significantly higher than currentlyknown processes.

[0030] Processes of the present invention for growing microorganisms ofthe order Thraustochytriales include adding a source of carbon and asource of nitrogen to a fermentation medium comprising themicroorganisms at a rate sufficient to increase the biomass density ofthe fermentation medium to those described above. This fermentationprocess, where a substrate (e.g., a carbon source and a nitrogen source)is added in increments, is generally referred to as a fed-batchfermentation process. It has been found that when the substrate is addedto a batch fermentation process the large amount of carbon sourcepresent (e.g., about 200 g/L or more per 60 g/L of biomass density) hada detrimental effect on the microorganisms. Without being bound by anytheory, it is believed that such a high amount of carbon source causesdetrimental effects, including osmotic stress, for microorganisms andinhibits initial productivity of microorganisms. Processes of thepresent invention avoid this undesired detrimental effect whileproviding a sufficient amount of the substrate to achieve the abovedescribed biomass density of the microorganisms.

[0031] Processes of the present invention for growing microorganisms caninclude a biomass density increasing stage. In the biomass densityincreasing stage, the primary objective of the fermentation process isto increase the biomass density in the fermentation medium to obtain thebiomass density described above. The rate of carbon source addition istypically maintained at a particular level or range which does not causea significant detrimental effect on productivity of microorganisms. Anappropriate range of the amount of carbon source needed for a particularmicroorganism during a fermentation process is well known to one ofordinary skill in the art. Preferably, a carbon source of the presentinvention is a non-alcoholic carbon source, i.e., carbon source thatdoes not contain alcohol. As used herein, an “alcohol” refers to acompound having 4 or less carbon atoms with one hydroxy group, e.g.,methanol, ethanol and isopropanol. More preferably, a carbon source ofthe present invention is a carbohydrate, including, but not limited to,fructose, glucose, sucrose, molasses, and starch. Other suitable simpleand complex carbon sources and nitrogen sources are disclosed in theabove-referenced patents. Typically, however, a carbohydrate, preferablycorn syrup, is used as the primary carbon source.

[0032] A particularly preferred nitrogen source is inorganic ammoniumsalt, more preferably ammonium salts of sulfate, hydroxide, and mostpreferably ammonium hydroxide.

[0033] When ammonium is used as a nitrogen source, the fermentationmedium becomes acidic if it is not controlled by base addition orbuffers. When ammonium hydroxide is used as the primary nitrogen source,it can also be used to provide a pH control. The microorganisms of theorder Thraustochytriales, in particular Thraustochytriales of the genusThraustochytrium and Schizochytrium, will grow over a wide pH range,e.g., from about pH 5 to about pH 11. A proper pH range for fermentationof a particular microorganism is within the knowledge of one skilled inthe art.

[0034] Processes of the present invention for growing microorganisms canalso include a production stage. In this stage, the primary use of thesubstrate by the microorganisms is not increasing the biomass densitybut rather using the substrate to produce lipids. It should beappreciated that lipids are also produced by the microorganisms duringthe biomass density increasing stage; however, as stated above, theprimary goal in the biomass density increasing stage is to increase thebiomass density. Typically, during the production stage the addition ofthe nitrogen substrate is reduced or preferably stopped.

[0035] It was previously generally believed that the presence ofdissolved oxygen in the fermentation medium is crucial in the productionof polyunsaturated compounds by eukaryotic microorganisms includingomega-3 and/or omega-6 polyunsaturated fatty acids. Thus, a relativelylarge amount of dissolved oxygen in the fermentation medium wasgenerally believed to be preferred. Surprisingly and unexpectedly,however, the present inventors have found that the production rate oflipids is increased dramatically when the dissolved oxygen level duringthe production stage is reduced. Thus, while the dissolved oxygen levelin the fermentation medium during the biomass density increasing stageis at least about 8% of saturation, and preferably at least about 4% ofsaturation, during the production stage the dissolved oxygen level inthe fermentation medium is reduced to about 3% of saturation or less,preferably about 1% of saturation or less, and more preferably about 0%of saturation. In one particular embodiment of the present invention,the amount of dissolved oxygen level in the fermentation medium isvaried during the fermentation process. For example, for a fermentationprocess with total fermentation time of from about 90 hours to about 100hours, the dissolved oxygen level in the fermentation medium ismaintained at about 8% during the first 24 hours, about 4% from about24^(th) hour to about 40^(th) hour, and about 0.5% or less from about40^(th) hour to the end of the fermentation process.

[0036] The amount of dissolved oxygen present in the fermentation mediumcan be controlled by controlling the amount of oxygen in the head-spaceof the fermentor, or preferably by controlling the speed at which thefermentation medium is agitated (or stirred). For example, a highagitation (or stirring) rate results in a relatively higher amount ofdissolved oxygen in the fermentation medium than a low agitation rate.For example, in a fermentor of about 14,000 gallon capacity theagitation rate is set at from about 50 rpm to about 70 rpm during thefirst 12 hours, from about 55 rpm to about 80 rpm during about 12^(th)hour to about 18^(th) hour and from about 70 rpm to about 90 rpm fromabout 18^(th) hour to the end of the fermentation process to achieve thedissolved oxygen level discussed above for a total fermentation processtime of from about 90 hours to about 100 hours. A particular range ofagitation speeds needed to achieve a particular amount of dissolvedoxygen in the fermentation medium can be readily determined by one ofordinary skill in the art.

[0037] A preferred temperature for processes of the present invention isat least about 20° C., more preferably at least about 25° C., and mostpreferably at least about 30° C. It should be appreciated that coldwater can retain a higher amount of dissolved oxygen than warm water.Thus, a higher fermentation medium temperature has additional benefit ofreducing the amount of dissolved oxygen, which is particularly desiredas described above.

[0038] Certain microorganisms may require a certain amount of salineminerals in the fermentation medium. These saline minerals, especiallychloride ions, can cause corrosion of the fermentor and other downstreamprocessing equipment. To prevent or reduce these undesired effects dueto a relatively large amount of chloride ions present in thefermentation medium, processes of the present invention can also includeusing non-chloride containing sodium salts, preferably sodium sulfate,in the fermentation medium as a source of saline (i.e., sodium). Moreparticularly, a significant portion of the sodium requirements of thefermentation are supplied as non-chloride containing sodium salts. Forexample, less than about 75% of the sodium in the fermentation medium issupplied as sodium chloride, more preferably less than about 50% andmore preferably less than about 25%. The microorganisms of the presentinvention can be grown at chloride concentrations of less than about 3g/L, more preferably less than about 500 mg/L, more preferably less thanabout 250 mg/L and more preferably between about 60 mg/L and about 120mg/L.

[0039] Non-chloride containing sodium salts can include soda ash (amixture of sodium carbonate and sodium oxide), sodium carbonate, sodiumbicarbonate, sodium sulfate and mixtures thereof, and preferably includesodium sulfate. Soda ash, sodium carbonate and sodium-bicarbonate tendto increase the pH of the fermentation medium, thus requiring controlsteps to maintain the proper pH of the medium. The concentration ofsodium sulfate is effective to meet the salinity requirements of themicroorganisms, preferably the sodium concentration is (expressed as g/Lof Na) at least about 1 g/L, more preferably in the range of from about1 g/L to about 50 g/L and more preferably in the range of from about 2.0g/L to about 25 g/L.

[0040] Various fermentation parameters for inoculating, growing andrecovering microorganisms are discussed in detail in U.S. Pat. No.5,130,242, which is incorporated herein by reference in its entirety.Any currently known isolation methods can be used to isolatemicroorganisms from the fermentation medium, including centrifugation,filtration, decantation, and solvent evaporation. It has been found bythe present inventors that because of such a high biomass densityresulting from processes of the present invention, when a centrifuge isused to recover the microorganisms it is preferred to dilute thefermentation medium by adding water, which reduces the biomass density,thereby allowing more effective separation of microorganisms from thefermentation medium.

[0041] Preferably, the microorganisms are recovered in a dry form fromthe fermentation medium by evaporating water from the fermentationmedium, for example, by contacting the fermentation medium directly(i.e., without pre-concentration, for example, by centrifugation) with adryer such as a drum-dryer apparatus, i.e., a direct drum-dryer recoveryprocess. When using the direct drum-dryer recovery process to isolatemicroorganisms, typically a steam heated drum-dryer is employed. Inaddition when using the direct drum-dryer recovery process, the biomassdensity of the fermentation medium is preferably at least about 130 g/L,more preferably at least about 150 g/L, and most preferably at leastabout 180 g/L. This high biomass density is generally desired for thedirect drum-dryer recovery process because at a lower biomass density,the fermentation medium comprises a sufficient amount of water to coolthe drum significantly, thus resulting in incomplete drying ofmicroorganisms. Other methods of drying cells, including spray-drying,are well known to one of ordinary skill in the art.

[0042] Processes of the present invention provide a lipid productionrate of at least about 0.5 g/L/hr, preferably at least about 0.7 g/L/hr,more preferably at least about 0.9 g/L/hr, and most preferably at leastabout 1.0 g/L/hr. Moreover, lipids produced by processes of the presentinvention contain polyunsaturated lipids in the amount greater thanabout 15%, preferably greater than about 20%, more preferably greaterthan about 25%, still more preferably greater than about 30%, and mostpreferably greater than about 35%. Lipids can be recovered from eitherdried microorganisms or from the microorganisms in the fermentationmedium. Generally, at least about 20% of the lipids produced by themicroorganisms in the processes of the present invention are omega-3and/or omega-6 polyunsaturated fatty acids, preferably at least about30% of the lipids are omega-3 and/or omega-6 polyunsaturated fattyacids, more preferably at least about 40% of the lipids are omega-3and/or omega-6 polyunsaturated fatty acids, and most preferably at leastabout 50% of the lipids are omega-3 and/or omega-6 polyunsaturated fattyacids. Alternatively, processes of the present invention provides a DHAproduction rate of at least about 0.2 g of DHA/L/hr, preferably at leastabout 0.3 g of DHA/L/hr, more preferably at least about 0.4 g ofDHA/L/hr, and most preferably at least about 0.5 g of DHA/L/hr. Stillalternatively, at least about 25% of the lipid is DHA (based on totalfatty acid methyl ester), preferably at least about 30% more preferablyat least about 35%, and most preferably at least about 40%.

[0043] Microorganisms, lipids extracted therefrom, the biomass remainingafter lipid extraction or combinations thereof can be used directly as afood ingredient, such as an ingredient in beverages, sauces, dairy basedfoods (such as milk, yogurt, cheese and ice-cream) and baked goods;nutritional supplement (in capsule or tablet forms); feed or feedsupplement for any animal whose meat or products are consumed by humans;food supplement, including baby food and infant formula; andpharmaceuticals (in direct or adjunct therapy application). The term“animal” means any organism belonging to the kingdom Animalia andincludes, without limitation, any animal from which poultry meat,seafood, beef, pork or lamb is derived. Seafood is derived from, withoutlimitation, fish, shrimp and shellfish. The term “products” includes anyproduct other than meat derived from such animals, including, withoutlimitation, eggs, milk or other products. When fed to such animals,polyunsaturated lipids can be incorporated into the flesh, milk, eggs orother products of such animals to increase their content of theselipids.

[0044] Additional objects, advantages, and novel features of thisinvention will become apparent to those skilled in the art uponexamination of the following examples thereof, which are not intended tobe limiting.

EXAMPLES

[0045] The strain of Schizochytrium used in these examples produces twoprimary polyenoic acids, DHAn-3 and DPAn-6 in the ratio of generallyabout 3:1, and small amounts of other polyenoic acids, such as EPA andC20:3, under a wide variety of fermentation conditions. Thus, whilefollowing examples only list the amount of DHA, one can readilycalculate the amount of DPA produced by using the above disclosed ratio.

Example 1

[0046] This example illustrates the affect of oxygen content in afermentation medium on lipid productivity.

[0047] Fermentation results of Schizochytrium ATCC No. 20888 at variouslevels of dissolved oxygen content were measured. The results are shownin FIG. 1, where RCS is residual concentration of sugar, and DCW isdry-cell weight.

Example 2

[0048] This example illustrates the reproducibility of processes of thepresent invention.

[0049] Microorganisms were produced using fermentors with a nominalworking volume of 1,200 gallons. The resulting fermentation broth wasconcentrated and microorganisms were dried using a drum-dryer. Lipidsfrom aliquots of the resulting microorganisms were extracted andpurified to produce a refined, bleached, and deodorized oil.Approximately 3,000 ppm of d-1-α-tocopheryl acetate was added fornutritional supplementation purposes prior to analysis of the lipid.

[0050] Nine fermentations of Schizochytrium ATCC No. 20888 were run andthe results are shown in Table 1. The dissolved oxygen level was about8% during the first 24 hours and about 4% thereafter. TABLE 1 Fed-batchfermentation results for the production of DHA. Age Entry (Hrs) Yield¹(g/L) DHA (%) FAME² (%) Productivity³ 1 100.3 160.7 17.8 49.5 0.285 299.8 172.4 19.4 51.3 0.335 3 84.7 148.7 14.4 41.4 0.253 4 90.2 169.519.7 53.9 0.370 5 99.0 164.1 12.5 38.9 0.207 6 113.0 187.1 19.7 47.20.326 7 97.0 153.5 13.7 41.0 0.217 8 92.8 174.8 16.4 48.6 0.309 Avg₄97.1 166.4 16.7 46.5 0.288 Std₅ 8.4 12.3 2.9 5.4 0.058 CV₆ (%) 8.7 7.417.3 11.7 20.2

[0051] Corn syrup was fed until the volume in the fermentor reachedabout 1,200 gallons, at which time the corn syrup addition was stopped.The fermentation process was stopped once the residual sugarconcentration fell below 5 g/L. The typical age, from inoculation tofinal, was about 100 hours.

[0052] The fermentation broth, i.e., fermentation medium, was dilutedwith water using approximately a 2:1 ratio to reduce the ash content ofthe final product and help improve phase separation during thecentrifugation step. The concentrated cell paste was heated to 160° F.(about 71° C.) and dried on a Blaw Knox double-drum dryer (42″×36″).Preferably, however, microorganisms are dried directly on a drum-dryerwithout prior centrifugation.

[0053] The analysis result of lipids extracted from aliquots of eachentries in Table 1 is summarized in Table 2. TABLE 2 Analysis of lipidsfrom microorganisms of Table 1. % DHA relative Total Lipid % Entry toFAME¹ by wt. 1 36.0 72.3 2 37.8 70.3 3 34.8 61.5 4 36.5 74.8 5 32.1 52.86 41.7 67.7 7 33.4 49.9 8 33.7 61.4 Avg 35.8 63.8 Std.₃ 3.0 9.1 CV₄ (%)8.5 14.2

[0054] Unless otherwise stated, the fermentation medium used throughoutthe Examples section includes the following ingredients, where the firstnumber indicates nominal target concentration and the number inparenthesis indicates acceptable range: sodium sulfate 12 g/L (11-13);KCl 0.5 g/L (0.45-0.55); MgSO₄.7H₂O 2 g/L (1.8-2.2); Hodag K-60 antifoam0.35 g/L (0.3-0.4); K₂SO₄ 0.65 g/L (0.60-0.70); KH₂PO₄ 1 g/L (0.9-1.1);(NH₄)₂SO₄ 1 g/L (0.95-1.1); CaCl₂.2H₂O 0.17 g/L (0.15-0.19); 95 DE cornsyrup (solids basis) 4.5 g/L (2-10); MnCl₂.4H₂O 3 mg/L (2.7-3.3);ZnSO₄.7H₂O 3 mg/L (2.7-3.3); CoCl₂.6H₂O 0.04 mg/L (0.035-0.045);Na₂MoO₄.2H₂O 0.04 mg/L (0-0.045); CuSO₄.5H₂O 2 mg/L (1.8-2.2);NiSO₄.6H₂O 2 mg/L (1.8-2.2); FeSO₄.7H₂O 10 mg/L (9-11); thiamine 9.5mg/L (4-15); vitamin B₁₂ 0.15 mg/L (0.05-0.25) and Ca_(1/2) Pantothenate3.2 mg/L (1.3-5.1). In addition, 28% NH₄OH solution is used as thenitrogen source.

[0055] The ash content of the dried microorganisms is about 6% byweight.

Example 3

[0056] This example illustrates the effect of reduced dissolved oxygenlevel in the fermentation medium on the productivity of microorganismsusing G-tank scale.

[0057] Using the procedure described in Example 2, a 14,000 gallonnominal volume fermentation was conducted using a wild-type strainSchizochytrium, which can be obtained using isolation processesdisclosed in the above mentioned U.S. Pat. Nos. 5,340,594 and 5,340,742.The dissolved oxygen level in the fermentation medium was about 8%during the first 24 hours, about 4% from the 24^(th) hour to the 40^(th)hour and about 0.5% from the 40^(th) hour to the end of fermentationprocess. Results of this lower dissolved oxygen level in fermentationmedium processes are shown in Table 3. TABLE 3 14,000 gallon scalefermentation of Schizochytrium. % DHA DHA Productivity Entry Age (Hrs)Yield (g/L) % DHA % FAME rel. to FAME (g of DHA/L/hr)  1 82.0 179.3 21.752.4 41.4 0.474  2 99.0 183.1 22.3 55.0 40.5 0.412  3 72.0 159.3 — —40.9 —  4 77.0 161.3 — — 43.2 —  5 100.0 173.0 23.9 53.3 44.9 0.413  6102.0 183.3 21.6 50.8 42.6 0.388  7 104.0 185.1 23.7 55.0 43.1 0.422  888.0 179.3 22.3 52.6 42.4 0.454  9 100.0 166.4 22.5 53.5 42.1 0.374 1097.0 182.6 22.8 51.6 44.1 0.429 11 87.5 176.5 19.8 45.6 43.5 0.399 1267.0 170.8 18.8 48.1 39.1 0.479 13 97.0 184.9 23.2 52.7 44.0 0.442 14102.0 181.9 23.6 52.9 44.6 0.421 15 102.0 186.9 19.9 47.8 41.8 0.365 1697.0 184.4 19.6 45.5 43.0 0.373 17 98.0 174.7 19.7 45.1 43.7 0.351 18103.5 178.8 18.3 44.5 41.2 0.316 19 102.0 173.7 15.8 43.1 36.7 0.269 2094.0 190.4 19.3 46.9 41.1 0.391 21 72.0 172.5 22.8 52.8 43.2 0.546 2275.0 173.1 21.0 51.7 40.8 0.485 23 75.0 152.7 20.3 50.3 40.4 0.413 2475.5 172.5 21.9 51.7 42.3 0.500 25 61.0 156.4 17.3 45.7 37.8 0.444 2674.5 150.6 20.2 50.1 40.2 0.408 27 70.5 134.3 14.8 40.6 36.6 0.282 2875.5 146.1 21.3 49.7 42.8 0.412 29 82.0 174.3 21.4 50.4 42.5 0.455 30105.0 182.3 21.7 50.7 42.8 0.377 31 66.0 146.2 16.4 44.6 36.7 0.363 Avg87.2 171.5 20.6 49.5 41.6 0.409 Std 13.9 14.1 2.4 3.8 2.3 0.061 CV 16.0%8.2% 11.6% 7.7% 5.5% 15.0%

Example 4

[0058] This example illustrates the effect of reduced dissolved oxygenlevel in the fermentation medium on the productivity of microorganismson a 41,000 gallon scale.

[0059] Same procedure as Example 3 in a 41,000 gallon fermentor wasperformed. Results are shown in Table 4. TABLE 4 41,000 gallon scalefermentation of Schizochytrium % DHA DHA Productivity Entry Age (Hrs)Yield (g/L) % DHA % FAME rel. to FAME (g of DHA/L/hr) 1 75.0 116.1 17.346.1 37.4 0.268 2 99.0 159.3 17.4 47.0 37.1 0.280 3 103.0 152.6 16.047.2 33.8 0.237 4 68.0 136.8 17.9 45.9 39.1 0.360 5 84.0 142.0 17.5 47.037.2 0.296 Avg 85.8 141.4 17.2 46.6 36.9 0.288 Std 15.1 16.6 0.7 0.6 1.90.046 CV 17.5% 11.8 4.2% 1.3% 5.2% 15.8%

Example 5

[0060] This example illustrates the affect of extra nitrogen on thefermentation process of the present invention.

[0061] Four sets of 250-L scale fed-batch experiments were conductedusing a procedure similar to Example 3. Two control experiments and twoexperiments containing extra ammonia (1.15× and 1.25× the normal amount)were conducted. Results are shown in Table 5. TABLE 5 Affects of extraammonia on fermentation of Schizochytrium. Conver- Age Yield Biomasssion DHA FAME DHA (hrs) (g/L) Productivity Efficiency Content ContentProductivity Sugar target: 7 g/L, Base pH set point: 5.5, Acid pH setpoint: 7.3, 1.0 × NH₃ 48 178 3.71 g/L/hr 51.5% 10.7% 37.8% 0.40 g/L/hr60 185 3.08 g/L/hr 46.9% 16.3% 47.2% 0.50 g/L/hr 72 205 2.85 g/L/hr45.2% 17.4% 47.4% 0.50 g/L/hr 84 219 2.61 g/L/hr 43.8% 17.1% 45.5% 0.45g/L/hr 90 221 2.46 g/L/hr 44.1% 18.4% 48.9% 0.45 g/L/hr Sugar target: 7g/L, Base pH set point: 5.5, Acid pH set point: 7.3, 1.15 × NH₃ 48 1713.56 g/L/hr 55.6% 12.0% 36.3% 0.43 g/L/hr 60 197 3.28 g/L/hr 54.6% 9.4%38.4% 0.31 g/L/hr 72 191 2.65 g/L/hr 52.8% 9.4% 40.0% 0.25 g/L/hr 84 1902.26 g/L/hr 52.5% 10.0% 42.5% 0.23 g/L/hr 90 189 2.10 g/L/hr 52.2% 9.2%43.3% 0.19 g/L/hr Sugar target: 7 g/L, Base pH set point: 5.5, Acid pHset point: 7.3, 1.25 × NH₃ 48 178 3.71 g/L/hr 56.4% 11.5% 33.7% 0.43g/L/hr 60 179 2.98 g/L/hr 48.6% 10.3% 36.0% 0.31 g/L/hr 72 180 2.50g/L/hr 48.8% 12.0% 37.6% 0.30 g/L/hr 84 181 2.15 g/L/hr 46.1% 13.6%40.1% 0.29 g/L/hr 90 185 2.06 g/L/hr 45.7% 12.6% 40.7% 0.26 g/L/hr Sugartarget: 7 g/L, Base pH set point: 5.5, Acid pH set point: 7.3, 1.0 × NH₃48 158 3.29 g/L/hr 55.7% 13.1% 36.5% 0.43 g/L/hr 60 174 2.90 g/L/hr48.9% 17.9% 39.2% 0.52 g/L/hr 72 189 2.63 g/L/hr 45.7% 21.0% 39.4% 0.55g/L/hr 84 196 2.33 g/L/hr 44.1% 22.4 40.1% 0.52 g/L/hr 90 206 2.29g/L/hr 44.8% 22.1% 40.3% 0.51 g/L/hr

[0062] In general, extra nitrogen has a negative effect on fermentationperformance, as significant reductions were observed in the DHAproductivity for the two batches where extra ammonia were added. Asshown on Table 5, the control batches resulted in final DHA levels of18.4% and 22.1% versus the 9.2% (1.15× ammonia) and 12.6% (1.25×ammonia) for extra nitrogen supplemented batches.

Example 6

[0063] This example shows a kinetic profile of a fermentation process ofthe present invention.

[0064] A 1000 gallon scale fed-batch experiment was conducted using aprocedure similar to Example 3. Kinetic profile of the fermentationprocess is shown in Table 6. TABLE 6 Kinetic Profile for a 1,000 gallonscale Fed-Batch fermentation of Schizochytrium. Age Yield BiomassConversion % DHA % FAME DHA (hrs) (g/L) Productivity Efficiency ContentContent Productivity 24 118 4.92 g/L/hr 78.2% 7.4 18.8 0.36 g/L/hr 30138 4.60 g/L/hr 60.3% 10.6 30.9 0.49 g/L/hr 36 138 3.83 g/L/hr 46.6%11.6 36.5 0.44 g/L/hr 42 175 4.17 g/L/hr 49.8% 13.4 41.7 0.56 g/L/hr 48178 3.71 g/L/hr 45.1% 18.7 52.8 0.69 g/L/hr  48* 164 3.42 g/L/hr 41.5%15.3 33.1 0.52 g/L/hr 54 196 3.63 g/L/hr 45.7% 16.6 51.2 0.60 g/L/hr 60190 3.17 g/L/hr 41.7% 16.9 33.9 0.54 g/L/hr 72 189 2.62 g/L/hr 39.1%15.6 31.8 0.41 g/L/hr 84 195 2.32 g/L/hr 38.5% 16.4 32.7 0.38 g/L/hr 90200 2.22 g/L/hr 39.0% 18.8 33.3 0.42 g/L/hr 90 171 1.90 g/L/hr 33.3%22.2 61.6  0.42 g/L/hr**

Example 7

[0065] This example illustrates affect of the amount of carbon source onproductivity.

[0066] Three different fermentation processed using the process ofExample 3 were conducted using various amounts of carbon source. Resultsare shown on Table 7. TABLE 7 Fermentation results for various amountsof carbon source on fermentation of Schizochytrium. Age Yield CarbonConversion % DHA % FAME Productivity (hrs) (g/L) Charge EfficiencyContent Content (g/L/hr) 90 171 51.3% 33.3% 22.2 61.6 0.42 94 122 40.5%30.1% 19.1 57.3 0.25 59 73 20.0% 36.5% 11.9 40.8 0.15

[0067] The present invention, in various embodiments, includescomponents, methods, processes, systems and/or apparatus substantiallyas depicted and described herein, including various embodiments,subcombinations, and subsets thereof. Those of skill in the art willunderstand how to make and use the present invention after understandingthe present disclosure. The present invention, in various embodiments,includes providing devices and processes in the absence of items notdepicted and/or described herein or in various embodiments hereof,including in the absence of such items as may have been used in previousdevices or processes, e.g., for improving performance, achieving easeand\or reducing cost of implementation.

[0068] The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

What is claimed is:
 1. A process for producing lipids containing polyenoic fatty acids from eukaryotic microorganisms capable of producing at least about 20% of their biomass as lipids comprising adding to a fermentation medium comprising said microorganisms a non-alcoholic carbon source and a nitrogen source at a rate sufficient to increase the biomass density of said fermentation medium to at least about 100 g/L.
 2. The process of claim 1, wherein said process comprises a biomass density increasing stage and a production stage, wherein said biomass density increasing stage comprises adding said carbon source and said nitrogen source and said production stage comprises adding said carbon source without adding said nitrogen source to induce nitrogen limiting conditions which induces lipid production.
 3. The process of claim 2, wherein the amount of dissolved oxygen present in said fermentation medium during said production stage is lower than the amount of dissolved oxygen present in said fermentation medium during said biomass density increasing stage.
 4. The process of claim 3, wherein the amount of dissolved oxygen in said fermentation medium during said biomass density increasing stage is at least about 4%.
 5. The process of claim 4, wherein the amount of dissolved oxygen in said fermentation medium during said production stage is less than about 1%.
 6. The process of claim 1, wherein said non-alcoholic carbon source comprises a carbohydrate.
 7. The process of claim 1, wherein said nitrogen source comprises an inorganic ammonium salt.
 8. The process of claim 7, wherein said nitrogen source comprises ammonium hydroxide.
 9. The process of claim 8, wherein pH of the fermentation medium is controlled by said nitrogen source.
 10. The process of claim 1, wherein said fermentation medium is at a temperature of at least about 20° C.
 11. The process of claim 1, wherein said process produces lipids at a rate of at least about 0.5 g/L/hr.
 12. The process of claim 11, wherein at least about 15% of said lipids are polyunsaturated lipids.
 13. The process of claim 11, wherein the total amount of omega-3 and omega-6 fatty acids is at least about 20% of said lipids.
 14. The process of claim 11, wherein at least about 25% of said lipids is docosahexaenoic acid.
 15. The process of claim 1, wherein said microorganisms are selected from the group consisting of algae, fungi, protists, and mixtures thereof, wherein said microorganisms are capable of producing polyenoic fatty acids or other lipids which requires molecular oxygen for their synthesis.
 16. The process of claim 15, wherein said microorganisms are capable of producing lipids at a fermentation medium oxygen level of about less than 3% of saturation.
 17. The process of claim 15, wherein said microorganisms are grown in a fed-batch process.
 18. The process of claim 17 further comprising maintaining an oxygen level of less than about 3% of saturation in said fermentation medium during second half of said fermentation process.
 19. The process of claim 1, wherein said microorganisms are algae.
 20. The process of claim 19, wherein said microorganisms are of the order Thraustochytriales.
 21. The process of claim 20, wherein said microorganisms are selected from the group consisting of Thraustochytrium, Schizochytrium, and mixtures thereof.
 22. A process for growing eukaryotic microorganisms capable of producing at least about 20% of their biomass as lipids, said process comprising adding to a fermentation medium comprising said microorganisms a carbon source and a nitrogen source at a rate sufficient to increase the biomass density of said fermentation medium to at least about 100 g/L, wherein said process produces at least about 0.5 g/L/hr of lipids, and wherein at least about 15% of the total lipids produced by said microorganisms is polyunsaturated lipids.
 23. The process of claim 22, wherein said process further comprises: (a) a biomass density increasing stage, wherein the dissolved oxygen level in the fermentation medium is at least about 4%; and (b) a high lipid production stage, wherein the dissolved oxygen level in the fermentation medium is less than about 1%.
 24. The process of claim 22, wherein said carbon source is a non-alcohol carbon source.
 25. The process of claim 22, wherein said nitrogen source comprises an inorganic ammonium salt.
 26. The process of claim 22, wherein said microorganisms are algae.
 27. The process of claim 26, wherein said microorganisms are of the order Thraustochytriales.
 28. The process of claim 27, wherein said microorganisms are selected from the group consisting of Thraustochytrium, Schizochytrium, and mixtures thereof.
 29. The process of claim 22, wherein the total amount of omega-3 and omega-6 fatty acids is at least about 20% of said lipids.
 30. The process of claim 22, wherein at least about 25% of said lipids is docosahexaenoic acid.
 31. A process for growing eukaryotic microorganisms comprising adding to a fermentation medium comprising said microorganisms a carbon source and a nitrogen source at a rate sufficient to increase the biomass density of said fermentation medium to at least about 100 g/L, wherein said microorganisms are selected from the group consisting of algae, fungi, protists and mixtures thereof, and wherein said microorganisms are capable of producing at least about 20% of their biomass as lipids comprising polyenoic fatty acids.
 32. The process of claim 31, wherein said process produces microbial lipids at a rate of least about 0.5 g/L/hr.
 33. The process of claim 32, wherein greater than about 15% of said lipids is polyunsaturated lipids.
 34. The process of claim 32, wherein the total amount of omega-3 and omega-6 polyunsaturated fatty acids is at least about 20% of said lipids.
 35. The process of claim 32, wherein at least about 25% of said lipids is docosahexaenoic acid.
 36. The process of claim 31, wherein said process produces at least about 0.2 g/L/hr of docosahexaenoic acid.
 37. The process of claim 31, wherein said carbon source is non-alcoholic.
 38. The process of claim 37, wherein said carbon source is a carbohydrate.
 39. The process of claim 31, wherein said nitrogen source comprises an inorganic ammonium salt.
 40. The process of claim 39, wherein said nitrogen source comprises ammonium hydroxide.
 41. A process for producing eukaryotic microbial lipids comprising: (a) growing eukaryotic microorganisms in a fermentation medium to increase the biomass density of said fermentation medium to at least about 100 g/L; (b) providing a fermentation condition sufficient to allow said microorganisms to produce said lipids; and (c) recovering said lipids, wherein greater than about 15% of said lipids are polyunsaturated lipids.
 42. The process of claim 41, wherein a dissolved oxygen level in said fermentation medium during said microorganism growing step is higher than the dissolved oxygen level in said fermentation medium during said lipid producing step.
 43. The process of claim 42, wherein the dissolved oxygen level in said fermentation medium during said microorganism growing step is at least about 4%.
 44. The process of claim 42, wherein the dissolved oxygen level in said fermentation medium during said lipid producing step is less than about 1%.
 45. The process of claim 41, wherein said microorganisms are algae.
 46. The process of claim 45, wherein said microorganisms are of the order Thraustochytriales.
 47. The process of claim 46, wherein said microorganisms are selected from the group consisting of Thraustochytrium, Schizochytrium, and mixtures thereof.
 48. The process of claim 41, wherein the total amount of omega-3 and omega-6 fatty acids is at least about 20% of said microbial lipids.
 49. The process of claim 41, wherein at least about 25% of said microbial lipids is docosahexaenoic acid.
 50. The process of claim 41, wherein said process produces docosahexaenoic acid at a rate of at least about 0.2 g/L/hr.
 51. The process of claim 41, wherein said lipid recovery step comprises: (d) removing water from said fermentation medium to provide dry microorganisms; and (e) isolating said lipids from said dry microorganisms.
 52. The process of claim 51, wherein said water removal step comprises contacting said fermentation medium directly on a drum-dryer without prior centrifugation.
 53. The process of claim 41, wherein said microorganism growing step comprises adding a carbon source and a nitrogen source.
 54. The process of claim 53, wherein said carbon source is non-alcoholic.
 55. The process of claim 54, wherein said carbon source comprises a carbohydrate.
 56. The process of claim 53, wherein said nitrogen source comprises an inorganic ammonium salt.
 57. The process of claim 53, wherein said nitrogen source comprises ammonium hydroxide.
 58. A process for producing eukaryotic microbial lipids comprising: (a) adding a non-alcoholic carbon source and a nitrogen source to a fermentation medium comprising said microorganisms; (b) providing conditions sufficient for said microorganisms to produce said microbial lipids; and (c) recovering said microbial lipids, wherein at least about 15% of said microbial lipids are polyunsaturated lipids.
 59. The process of claim 58, wherein the dissolved oxygen level in said fermentation medium during a biomass density increasing step is higher than the dissolved oxygen level in said fermentation medium during said lipid producing step.
 60. The process of claim 59, wherein the dissolved oxygen level in said fermentation medium during said biomass density increasing step is at least about 4%.
 61. The process of claim 59, wherein the dissolved oxygen level in said fermentation medium during said lipid producing step is about 1% or less.
 62. The process of claim 58, wherein said microorganisms are selected from the group consisting of algae, protists, fungi and mixtures thereof.
 63. The process of claim 62, wherein said microorganisms are algae.
 64. The process of claim 63, wherein said microorganisms are of the order Thraustochytriales.
 65. The process of claim 64, wherein said microorganisms are selected from the group consisting of Thraustochytrium, Schizochytrium, and mixtures thereof.
 66. The process of claim 58, wherein said process produces said microbial lipids at a rate of at least about 0.5 g/L/hr.
 67. The process of claim 66, wherein the total amount of omega-3 and omega-6 fatty acids is at least about 20% of said microbial lipids.
 68. The process of claim 58, wherein at least about 25% of said lipids is docosahexaenoic acid.
 69. The process of claim 58, wherein said process produces docosahexaenoic acid at a rate of at least about 0.2 g/L/hr.
 70. The process of claim 58, wherein said lipid recovery step comprises: (d) removing water from said fermentation medium to provide dry microorganisms; and (e) isolating said lipids from said dry microorganisms.
 71. The process of claim 70, wherein said water removal step comprises contacting said fermentation medium directly on a drum-dryer without prior centrifugation.
 72. The process of claim 58, wherein said non-alcoholic carbon source comprises a carbohydrate.
 73. The process of claim 58, wherein said nitrogen source comprises an inorganic ammonium salt.
 74. The process of claim 58, wherein said nitrogen source comprises ammonium hydroxide.
 75. A process for producing eukaryotic microbial lipids comprising: (a) adding a carbon source and a nitrogen source to a fermentation medium comprising eukaryotic microorganisms and providing conditions sufficient to maintain a dissolved oxygen level of at least about 4% in said fermentation medium to produce a biomass density of at least about 100 g/L; (b) providing conditions sufficient to maintain the dissolved oxygen level of about 1% or less in said fermentation medium and providing conditions sufficient to allow said microorganisms to produce said lipids; and (c) recovering said microbial lipids, wherein at least about 15% of said microbial lipids are polyunsaturated lipids.
 76. The process of claim 75, wherein said microorganisms are selected from the group consisting of fungi, algae, protists and mixtures thereof.
 77. The process of claim 76, wherein said microorganisms are algae.
 78. The process of claim 77, wherein said microorganisms are of the order Thraustochytriales.
 79. The process of claim 78, wherein said microorganisms are selected from the group consisting of Thraustochytrium, Schizochytrium, and mixtures thereof.
 80. The process of claim 75, wherein at least about 20% of said microbial lipids are omega-3 and/or omega-6 fatty acids.
 81. The process of claim 75, wherein at least about 25% of said microbial lipids are docosahexaenoic acid.
 82. The process of claim 75, wherein said process produces docosahexaenoic acid at a rate of at least about 0.2 g/L/hr.
 83. The process of claim 75, wherein said lipid recovery step comprises: (d) removing water from said fermentation medium to provide dry microorganisms; and (e) isolating said lipids from said dry microorganisms.
 84. The process of claim 83, wherein said water removal step comprises contacting said fermentation medium directly on a drum-dryer without prior centrifugation. 